Double labeling immunoelectron microscopic study on the synaptic connections between glutamic acid neurons and GABA neurons in the hippocampus of rats

Summary In order to explore the roles of different neurotransmitters in epileptic pathogenesis, the synaptic connections between glutamic acid (Glu) neurons and GABA neurons in normal rat hippocampus were studied by pre-embedding double labeling immunoelectron microscopy. The GABA immunoreaction was first demonstrated by chromogen DAB, then the Glu immunoreaction was demonstrated by molybdic acid-TMB method. After being stabilized by DAB-cobalt chloride, the sections were processed for electron microscopic embedding. Under electron microscope, there were many Glu immunoreaction-positive neurons in the pyramidal layer of hippocampal CA₁ area and some GABA immunoreaction-positive neurons with pyramidal or polygonal perikarya in the pyramidal, polymorphic and radiant layer of CA₁ area. There were also symmetric dendro-axonic synapses formed by GABA-positive dendrites and Glu-positive axons in the polymorphic layer and symmetric axo-dendritic synapses formed by GABA-positive axons and Glu-positive dendrites in the radiant layer. In addition, there were symmetric autoregulatory axo-dendritic synapses between Glu-positive axons and dendrites and autoregulatory axo-axonic synapses (both symmetric and asymmetric) between GABA-positive axons. Above mentioned results, for the first time, showed that there were complex synaptic regulatory relationships between excitatory Glu neurons and inhibitory GABA neurons in the hippocampal CA₁ area, thereby, providing ultrastructural evidence for different neurotransmitters participating in epileptic pathogenesis. This project was supported by a grant from the National Nature Scientific Foundation of China (No. 39330210).     

电惊厥大鼠海马生长抑素神经元的超微结构变化

观察电惊厥大鼠海马生长抑素神经元的超微结构变化，为探讨生长抑素在癫痫发病中的作用机制提供形态学依据。采用电惊厥癫痫动物模型和免疫电镜方法观察。结果：电惊厥时海马内生长抑素神经元胞体和突起出现不同程度的结构损伤，表现为线粒体肿胀、细胞器崩解和神经末梢变性等；在海马区多形细胞层和分子层生长抑素神经元胞体、突起与非生长抑素神经元胞体、突起之间有复杂的突触联系。结论：电惊厥时海马CA4生长抑素神经元结构发生损伤。     

大鼠孤束核内含NT神经元的超微结构观察

用免疫电镜法对大鼠孤束核内侧亚核内NT-LI神经元的超微结构进行了观察。NT-LI产物可见于核周体、树突、轴突及终末内。标记轴突与树突构成轴-树突触最多见,轴-轴和轴-体突触少见。可见一些复合型突触和轴-树型自调节突触。此外一些NT-LI树突紧邻毛细血管。结果提示:(1)NT在孤束核调节心血管内脏活动中可能起着重要作用。(2)孤束核内NT神经元与血管之间可能存在神经-血管回路。     

小鼠海马内HDAC2的表达及其与NMDA受体、PSD-95的共定位

目的探讨组蛋白去乙酰化酶2(HDAC2)在成年C57BL/6小鼠海马内的分布及其与突触后致密区(PSD)蛋白成员的共定位,为揭示HDAC2与PSD蛋白复合物之间的内在联系及在海马相关的学习记忆过程中可能起到的调控作用提供形态学依据。方法应用免疫组化方法观察HDAC2在C57BL/6小鼠海马各区的表达分布。应用免疫荧光双标技术研究HDAC2与PSD蛋白成员N-甲基-D-天冬氨酸(NMDA)受体亚单位1(NR1)、PSD-95之间是否存在共定位。结果 HDAC2在小鼠海马CA1～CA3区锥体细胞和齿状回颗粒细胞均具有明显表达,而在各区的始层、辐射层、腔隙-分子层以及齿状回多形细胞层表达均较少。免疫荧光双标染色图片的重叠表明,HDAC2与NR1、PSD-95在小鼠海马CA1～CA3区锥体细胞层和齿状回颗粒细胞层内均可见显著共表达现象,其他区域偶见散在分布的双染神经元。结论 HDAC2在小鼠海马锥体细胞层和颗粒细胞层表达丰富,并与PSD蛋白成员间存在共定位现象。本实验结果为探讨HDAC2对谷氨酸能突触后神经元依赖的突触可塑性的调节机制提供了形态学依据。     

马桑内酯在大鼠离体海马脑片上引起的癫痫样放电活动

Coriaria lactone (CL) is an active constituent of a medicinal herb used as psychosolytic in traditional Chinese medicine. Recently it has been found that CL appears to act as a convulsant agent. Subsequently both acute and chronic epilepsy models caused by CL have been established successfully. In order to observe further the epileptogenic effects of CL in vitro, the hippocampal slice technique was used in experiments with 36 slices. The results are as follows: Addition of CL to the perfusion bathing the slices of rat hippocampus increased evoked-response in body layer of CA1 in a dose-dependent manner, and induced epileptiform burst. CL augmented population spike of CA1 pyramidal neurons triggered by either orthodromic stimulus (through synapses) or antidromic stimulus (direct effect on the axon of CA1 pyramidal cells) without alteration of presynaptic fiber volley and field-EPSP, and there was no significant change in latency of burst. Finally GABA had a weak effect on CL-induced epileptiform activity. These observations suggest that CL probably has a direct effect on the soma of pyramidal neurons, CL-induced epileptiform burst may be a result of altering the innate capacity of burst and increasing the intrinsic excitability of pyramidal cell membranes.     

利多卡因对大鼠海马锥体神经元GABA_A-Cl~-电流的影响

目的　研究利多卡因对大鼠海马锥体神经元全细胞γ 氨基丁酸介导的氯电流 (GABA Cl-电流 )的影响 ,及其中枢致惊厥作用的可能机制。方法　酶解急性分离 2周龄左右大鼠海马锥体神经元 ,采用全细胞膜片钳技术 ,记录利多卡因对单个海马锥体神经元GABA Cl-电流的影响。结果　将钳制电位固定在 - 6 0mV ,GA BA以剂量依赖性方式诱导出内向电流 ;利多卡因明显抑制GABA诱导的Cl-电流 (IGABA) ,5 0 %抑制浓度 (IC50 )为 4 .0 5mmol/L ,10mmol/L可使GABA浓度效应曲线明显右移 ,5 0 %有效浓度 (EC50 )由 7.72mmol/L增加到17.2mmol/L ,且最大电流明显降低。结论　利多卡因以非竞争性的方式 ,抑制海马锥体神经元GABAA Cl-电流 ,可能是其中枢致惊厥作用的机制。     

大鼠海马CA3区神经元和星形胶质细胞三维结构重塑

目的 观察大鼠海马CA3区神经元锥体细胞及其周围星形胶质细胞(AST)的分布,重塑两者之间的三维构象。方法 采用脑片膜片钳全细胞记录、细胞内荧光黄(LY)染色、免疫荧光和激光共聚焦显微镜检查(LSCM)相结合的技术。结果 根据放电形式的不同把海马锥体细胞分为位相型和非位相型放电神经元。LSCM下单层光学图像和三维立体重建显示许多AST紧密围绕在LY染色锥体细胞周围并形成紧密接触。2类神经元与AST形成接触的部位存在区别。非位相型放电神经元的树突和胞体周围都有许多AST形成接触的部位在,而位相型放电神经元则仅位于树突。结论 不同特性海马神经元周围AST的空间分布可能存在差异。     

大鼠海马CA3区神经元和星形胶质细胞三维结构重塑

目的 观察大鼠海马CA3区神经元锥体细胞及其周围星形胶质细胞(AST)的分布，重塑两者之间的三维构象。方法 采用脑片膜片钳全细胞记录、细胞内荧光黄(LY)染色、免疫荧光和激光共聚焦显微镜检查(LSCM)相结合的技术。结果 根据放电形式的不同把海马锥体细胞分为位相型和非位相型放电神经元。LSCM下单层光学图像和三维立体重建显示许多AST紧密围绕在LY染色锥体细胞周围并形成紧密接触。2类神经元与AST形成接触的部位存在区别。非位相型放电神经元的树突和胞体周围都有许多AST形成接触的部位在，而位相型放电神经元则仅位于树突。结论 不同特性海马神经元周围AST的空间分布可能存在差异。     

β2-nAChR促进小鼠海马CA1和CA3锥体神经元GABAA受体的功能成熟

目的探讨β2-烟碱型乙酰胆碱受体（β2-nAChR）在海马CA1和CA3锥体神经元的A型γ-氨基丁酸受体（GABAA-R）发育中 的作用。方法应用β2-nAChR基因敲除小鼠（β2-KO组）制备急性分离的海马CA1和CA3锥体神经元,应用穿孔膜片钳记录技 术记录GABAA-R选择性激动剂蝇蕈醇在CA1和CA3锥体神经元诱导的GABA电流,测试其平衡电位（EMus）和动力学指标,并 与野生型小鼠（WT组）进行比较。结果β2-KO组小鼠（n=4）CA1锥体神经元（n=7）的EMus为-31.7±3.5 mV,与WT组相比向去极 化偏移（P<0.05）;CA3锥体神经元（n=4）的EMus为-16.1±4.6 mV,同样较WT组偏向去极化方向（P<0.01）;与WT组小鼠不同,β2- KO组小鼠CA3和CA1神经元的EMus差异有统计学意义（P<0.05）。β2-KO组小鼠CA1和CA3神经元上都显示GABAA-R的失 敏显著减慢,衰减时间分别为2.2±0.2 s、3.2±0.1 s（WT组为1.6±0.1 s、2.3±0.1 s,P<0.05或P<0.01）。结论含β2的nAChR可能参 与促进小鼠海马CA1和CA3锥体细胞中GABAA-R的功能成熟。     

A Study of Epileptiform Hippocampal Unit Activity Induced by Intracerebroventricular Injection of Kainic Acid in Rats

Hippocampal EEG and unit activities were recorded after seizures were initiated withintracerebroventricular injection of kainic acid (KA) in rats. Three kinds of hippocampal unit re-sponses to KA were found:1. Positive units: These units were characterized by high frequency burst firing temporally coincidentwith each hippocampal EEG spike.2. Negative units: These units showed a cessation of firing during each EEG paroxysm.3. Indifferent units: These units showed no evident chanses coincident with EEG paroxysms.Most positive units were hippocampal complex spike cells which correspond histologically tohippocampal pyramidal cells, and most complex spike cells fired in positive bursts after KA treat-ment. In the early period after KA injection, the positive units were concentrated in CA3 area. It was suggested that the activities of positive units may be considered as the typical epileptiformhippocampal unit activity induced by KA, and the firing features of negative units ma be the resultof the influence of hippocampal inhibitory interneurons or the result of excessive cellulardepolarization, and that hippocampal pyramidal cells were more sensitive to the epileptogenic ef-fect of KA than hippocampal intemeurons, and some pyramidal cells in CA3, in particular,may serve as "epileptic pacemaker neurons " in KA-induced epileptogenesis.     

DHPG和BMI诱导的癫痫样放电模型的建立及比较

目的 在离体大鼠海马脑片上,通过激活Ⅰ型代谢型谷氨酸受体(mGluR)或阻断γ-氨基丁酸(GABA)受体,诱导癫痫样放电.方法 将离体大鼠海马脑片暴露于Ⅰ型mGluR特异性激动剂对羟基苯甘氨酸(DHPG)或GABAA受体阻断剂荷包牡丹碱(BMI),利用全细胞记录模式记录海马CA3区域单个锥体细胞的电活动.结果 暴露于DHPG或BMI中的海马脑片CA3区域的锥体细胞均产生癫痫样放电,且两者的放电频率比较差异无统计学意义(P>0.05).结论 在离体情况下,破坏神经兴奋性和抑制性系统的平衡可以诱导产生癫痫样放电活动.     

血管性痴呆大鼠海马CA1区神经元超微结构的长时程变化

目的观察血管性痴呆（VD）大鼠长时期海马组织学和CA1区神经元超微结构的动态变化规律。方法60只大鼠随机分为假手术组、模型7d组、模型15d组、模型1m组、模型2m组和模型4m组,每组10只。光镜和电镜下观察各组大鼠海马组织学和CA1区超微结构的改变。结果①光镜下假手术组大鼠海马CA1区未见明显病理变化,但模型组大鼠海马CA1区锥体细胞数量减少,细胞变性坏死,细胞明显水肿,排列紊乱;细胞核体积变小,深染,呈核固缩表现,顶树突缩短,排列紊乱。另外,模型1m组可见胶质细胞明显增生,模型2m组和4m组胶质细胞明显增生形成结节,呈现海马硬化表征。②电镜下可见神经细胞及神经末梢水肿、胞浆内有色素沉积、细胞膜断裂、核染色质边集、细胞核溶解、核周隙增宽,线粒体肿胀,嵴紊乱断裂,破坏严重。随造模时间的延长,病理改变逐渐加重。结论血管性痴呆发生和发展中海马CA1区锥体细胞严重脱失,神经元变性坏死,超微结构受损,并逐渐加重。     

皮质后索核投射纤维终末的超微结构和突触联系

本文采用顺行溃变法对猫后索核内皮质后索核投射纤维终末的超微结构和突触联系进行了研究。在电损毁皮质２～５天后，电镜下发现后索核内皮质后索核纤维终末存在三种溃变形式：即电子致密型、神经微丝型和电子透明型，以电子致密型最为多见。溃变的纤维终末常较粗大，主要含有大量密集的圆形突触小泡，主要与神经元的树突或胞体接触形成不对称型的轴－树突触、轴－轴突触、轴－体突触、轴－树－树系列突触，并参与形成以树突为中心的突触复合体。     

GABAA受体激动剂和拮抗剂对大鼠梨形皮质、海马Fos表达的影响及其与癫痫发病的关系

为探讨γ－氨基丁酸受体（ＧＡＢＡＡ）与癫痫发病机制之间的关系,应用免疫组化技术结合脑电图就ＧＡＢＡＡ受体激动剂蝇蕈醇对马桑内酯致痫大鼠大脑皮质、海马Ｆｏｓ表达的影响进行研究,并与ＧＡＢＡＡ受体拮抗剂荷包牡丹碱致痫大鼠的Ｆｏｓ表达进行比较。结果显示：侧脑室注射马桑内酯诱发癫痫发作后１ｈ,双侧齿状回颗粒细胞层、海马锥体细胞层及注射侧梨形皮质有大量Ｆｏｓ阳性细胞,对侧梨形皮质未检测到Ｆｏｓ表达;侧脑室注射蝇蕈醇可明显抑制齿状回、海马Ｆｏｓ的表达,但梨状皮质Ｆｏｓ表达未受影响。一侧侧脑室注射荷包牡丹碱诱发癫痫发作后１ｈ,双侧梨状皮质有较强的Ｆｏｓ表达,但齿状回、海马未见表达。脑电图也证实马桑内酯及荷包牡丹碱均能诱发棘慢波、尖慢波,蝇蕈醇则能明显抑制这种痫样放电。结果提示,ＧＡＢＡＡ受体在癫痫发病过程中起重要作用     

Whole-cell recordings of voltage-gated Calcium, Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons

Objective:To record Calcium, Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons. Methods:Hip-pocampal CA3 neurons were freshly isolated by 1 mg protease/3 ml SES and mechanical trituration with polished pipettes of progressively smaller tip diameters. Patch clamp technique in whole-cell mode was employed to record voltage-gated channel currents. Results:The procedure dissociated hippocampal neurons, preserving apical dendrites and several basal dendrites, without impairing the electrical characteristics of the neurons. Whole-cell patch clamp configuration was successfully used to record voltage-gated Ca2+ currents, delayed rectifier K+ current and voltage-gated Na+ currents. Conclusion:Protease combined with mechanical trituration may be used for the dissociation of neurons from rat hippocampus. Voltage-gated channels currents could be recorded using a patch clamp technique.     

马桑内酯在大鼠离体海马脑片上引起的癫痫样放电活动

采用大鼠离体海马脑片技术,建立了马桑内酯(Coriaria lactone,CL)引起的海马CA 1区锥体细胞癫痫样放电活动模型,并初步探讨了CL的致痫作用机理,可能与直接影响锥体细胞固有爆发放电能力有关。     

氯胺酮对离体海马神经元的浓度相关性作用

目的　了解氯胺酮对海马神经元的作用以分析其“分离麻醉”的机制。方法　采用常规细胞内记录技术 ,观察不同浓度的氯胺酮对成年大鼠海马CA1区锥体神经元的影响。结果　给予 0 .3、1及 3mmol/L的氯胺酮灌流 15min(n =16 ) ,能浓度依赖地增大细胞的输入斜率电阻、延长时间常数 (P <0 .0 5 ) ,但是 3mmol/L氯胺酮还降低了直接动作电位的幅度 (P <0 .0 5 )。氯胺酮对I F曲线及阈电位的影响显示 (n =12 ) :0 .3和 1mmol/L的氯胺酮分别使 2 5 %的细胞升高了内在兴奋性 ,而 1mmol/L还使 75 %的细胞降低了兴奋性 ,3mmol/L则降低了所有记录细胞的兴奋性。刺激Schaffer侧支在CA1锥体细胞诱发的兴奋性突触后电位 ,当灌流 0 .3mmol/L氯胺酮时有 80 %的细胞被增强 ;1mmol/L时有 91%的细胞出现先增强后抑制的作用 ,但在 3mmol/L时在所有的细胞反应被完全取消。结论　氯胺酮对海马CA1区锥体神经元具有低浓度兴奋而高浓度抑制的双向作用 ,可能是通过不同的机制完成的     

清醒大鼠侧脑室内注入红藻氨酸引起海马单位癫痫样电活动的研究

清醒大鼠侧脑室内注入红藻氨酸后,在海马内发现了正性、负性及无关单位等三种单位电反应。大多数正性单位来源于组织学上与海马锥体细胞相对应的海马复合峰电位细胞;同时,大多数海马复合峰电位细胞在注入红藻氨酸后表现为正性单位的放电形式。在注入红藻氨酸后的早期,正性单位主要集中于CA_3区。本文推测;(1)正性单位可能是红藻氨酸引起海马神经元癫痫样电活动的典型表现;负性单位的放电特征可能是其受海马中间抑制性神经元活动影响或是其过度去极化的结果;(2)海马内的锥体细胞比中间神经元对红藻氨酸的致癫作用更为敏感,CA_3区内的某些锥体细胞可能在红藻氨酸致癫过程中起着“起搏细胞”的作用。     

慢性应激所致大鼠海马长时程增强和氨基酸类神经递质的改变及苯妥英钠的效应

目的探讨慢性应激对大鼠海马长时程增强(LTP)和氨基酸类神经递质的影响及苯妥英钠对它们的效应.方法将24只SD雄性大鼠随机分为对照组、应激+生理盐水组和应激+苯妥英钠组,每组8只.采用离体海马脑片结合电生理的方法观测海马CA1区LTP的变化.以群体峰电位(PS)的幅值和场兴奋性突触后电位(fEPSP)的斜率作为观察LTP变化的指标.应用高效液相色谱紫外检测法检测海马氨基酸类神经递质的含量.结果(1)应激+生理盐水组PS幅值和fEPSP斜率在高频串刺激后增大的幅度低于对照组和应激+苯妥英钠组(P<0.05).(2)应激+生理盐水组和应激+苯妥英钠组的天冬氨酸含量[分别为(4.746±0.609)μmol/g和(4.948±0.751)μmol/g]高于对照组[(2.425±0.211)μmol/g,P<0.01];应激+生理盐水组的谷氨酸含量[(8.094±1.035)μmol/g]高于对照组[(6.016±0.677)μmol/g]和应激+苯妥英钠组[(6.970±0.647)μmol/g];P<0.05;应激+苯妥英钠组的GABA含量[(5.142±0.662)μmol/g]高于对照组[(4.229±0.449)μmol/g]和应激+生理盐水组[(4.249±0.463)μmol/g],P<0.01.结论慢性应激使大鼠海马CA1区LTP的形成受抑制,天冬氨酸和谷氨酸水平升高,GABA含量无明显改变;而苯妥英钠使慢性应激大鼠海马CA1区LTP维持正常.